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Reconstruction
Display image description
Medium spiny neuron from the mouse nucleus accumbens injected with Lucifer Yellow and Alexa 568 and reconstructed from a 3D opcial section series. The Alexa568 channel is shown here. The original data was acquired as a tiled mosaic that was stitched together in X-Y.
Full resolution image description
Multi-image tiff file (~570 Mb) containing the optical section series from the tiled mosaic. Note that the name of the file (Proj_EricsMontage.tif) is not the same as the microscopy product basename (acc1).
Volume_dimension
1440, 1782, 233
Volume scale
0.119, 0.119, 0.5
Animation description
rotation loop of maximum intensity projection of spiny neuron in nucleus accumbens. Some dendrites are incomplete due to the thickness of the section.

Segmentation
Display image description
Segmentation of dendritic tree from medium spiny neuron from the nucleus accumbens of the mouse using Neurolucida. Portions of the dendrite in which the spines were too dim to trace are indicated byt the gray *.
Segmentation file description
Output of Neurolucida tracing program in ascii format (acc1.spines3c.asc).

License
Attribution Only: This image is licensed under a Creative Commons Attribution License. View License Deed | View Legal Code

CCDB:1*  Cite 
Project: P0000
Project name
Mouse BIRN test data
Description
NCMIR test data for Mouse BIRN
Funding agency
NIH
Leader(s)
Maryann Martone
Collaborator(s)
Eric Bushong
Start date
09-01-2001
End date
unspecified
 
Experiment
Experiment ID
1
Experiment date
12-13-2001
Title
Intracellular Injection of Nucleus Accumbens Neuron
Purpose
to obtain multi resolution data for Mouse BIRN
Experimenter(s)
Eric Bushong
Microscopy product
Microscopy product ID
1
Instrument
BioRad MRC 1024 Confocal
Microscopy type
multiphoton
Product type
Optical section series and mosaic
Image basename
ACC1
Spatial Axis Image Size Pixel Size
X 512px 0.119 µm
Y 480px 0.119 µm
Subject
Species
mouse
Scientific name
mus musculus
Strain
C57BL/6
Treatment
none
Age
2 months
Age class
adult
Tissue section
Anatomical location
neostriatum
Microtome
vibratome
Thickness
100 µm
Specimen description
Organ
brain
System
central nervous system
Structure
dendritic tree
Cell type
medium spiny neuron
Atlas coordinate
0, 0, 0,
Imaging parameters
Type
Light microscopy product
Immersion medium
water
Mounting medium
water
Lens magnification
X
Refractive index
1
Specimen preparation
Protocol used
Photo-oxidation of Lucifer Yellow Injected Cells1) Anesthetize rat with ketamine cocktail solution (see below) using 0.4 mL/100 g body weight or Nembutal (0.1mL/100g).To prepare the ketamine cocktail solution:ketamine3.75 mlacepromazine0.30 mlrompun/xylazine1.90 mlsterile saline 23.0 ml Store in an airtight and light protected bottle for up to 3 months.2) Clear vasculature using Ringer's solution (37C):9.9 mL NaCl (79.8 g/L)1 mL KCl (37.5 g/L)1 mL Na2HPO4 (18 g/L)1 mL MgCl2 . 6 H20 (20.0g/L)2.5 mL NaHCO3 (50.0 g/L)Fill to 95.5 mL using ddH2O Warm to 37 C and bubble w/carbogen1 mL CaCl2 . 2 H2O (30.0 g/L)200 mg dextrose2.5 mL heparin1 mL xylocaine3) Perfuse with fixative for 6-15 minutes, depending on size of animal (6 minutes - mouse; 10 minutes - small rat; 15 minutes - large rat) using 4% paraformaldehyde in 0.1 M PBS, pH 7.4 (200 mL) at 37C:Heat 100 mL ddH2O to 60CAdd 8 g "Prill" paraformaldehydeAdd 4-6 drops 1 N NaOHFilter with #1 Whatman filterAdd 40 mL 5x PBSDilute to final volume of 200 mLAdd 0.1% glutaraldehyde if intending to photoconvert specimens. 4) Extract brain. If it is still soft, place in same fixative as above and allow to post-fix for 0.5-1 hour at 4C. Cut into 100-150m slices with Vibratome using a slow speed and the lowest frequency which will allow for proper cutting.5) Store slices in 0.1 M PBS in refrigerator. Slices should be used same day if planning to photoconvert. Given good initial fixation, slices will be usable for dye-filling for 2-3 days.6) Visualize tissue using IR-DIC Nomarski imaging. If necessary, counterstain tissue in 5 ?M acridine orange in 0.1 M PBS for 30 seconds. Microinject cells using 5% Lucifer Yellow-CH (lithium salt) in ddH2O. Use a positive retention current to prevent leakage, if necessary, and a negative pulsed current of 1-3 nA to inject cells. Allow cells to fill until all processes are equally fluorescent. 7) Place slices containing filled cells into 4% LY at 4C for at least 20-30 minutes. 8) Acquire light-level image of cell, using Mat-Tek dish to hold specimen. Mat-Tek dishes should be prepared ahead of time by treating with polyethyleneimine solution (0.1% aq.) for 30 sec., followed by brief rinse in ddH2O. Allow dish to dry and store in fridge until used. Can use PBS bubbled with Ar gas to try to reduce fading.9) Bubble DAB/potassium cyanide solution with O2: 2 mL DAB (10mg/mL)13 mg potassium cyanide2.66 mL 5x PBS 8.67 mL dd H2O9) Fix slice in 2% glutaraldehyde for 15-20 minutes:0.8 mL 25% glutaraldehyde2 mL 5x PBS7.2 mL dd H2O10) Wash 2-3 times in PBS.11) Place 100 mM glycine on slice for 1-2 minutes:38 mg glycine10 mL PBS12) Wash with PBS.13) Incubate slice in DAB/potassium cyanide solution for 8-10 min.14) Photo-oxidize slice with 75 W xenon lamp. Exchange DAB/potassium cyanide solution every couple of minutes. Allow reaction to proceed until all fluorescence is gone and brown reaction product is visible (about 10 minutes).15) Wash 3x 10 minutes in 0.1 M PBS at 4C.Conventional Embedding Procedure1) Post-fix in 0.5% OsO4 in 0.1 M PBS for 30 minutes at 4C.2) Rinse 3x 2 minutes in ddH2O at 4C.3) Dehydrate as follows: 70% EtOH, 10 minutes; 80% EtOH, 10 minutes; 90% EtOH, 10 minutes; 95% EtOH, 10 minutes; 100% EtOH, 2x 10 minutes; dry acetone, 2x 10 minutes (2nd time at room temp.)4) Infiltrate tissue in 50:50 acetone:DurcupanACM for 1 hour (or overnight).5) 100% resin for 2x 1 hour (2nd time fresh resin).6) Mount tissue on mould release slides and place in vacuum oven at 60C for 2 days.Microwave-Enhanced Embedding Procedure1) Osmium fix tissue in 600 L of 1% OsO4 for 2x 40 seconds. Solution should begin at < 10C and attain a final temp. of 30-35C. Microcentrifuge tube should not be placed in water bath during irradiation. 2) Rinse samples ddH20 for 2 minutes at room temp.3) Dehydration:50% EtOH2 X 40 sec.35C70% EtOH2 X 40 sec.35C90% EtOH2 X 40 sec.35C100% EtOH2 X 40 sec.35CDry Acetone2 X 40 sec.35CDehydration steps performed in water bath. Tubes should be filled with 600 L of solution for each step.4) Infiltration:1:1 Resin:acetone1 X 15 min.50C100% Resin3 X 10 min.50CFill bath with stock acetone and check level often to ensure that temp. probe is immersed in bath.5) Polymerization:Mount tissue on mould release slides and place in vacuum oven at 60C for 2 days.Obtain transmitted light z-series photo-oxidized, if desired, before sectioning. Photo-oxidation of Lucifer Yellow Injected Cells1) Anesthetize rat with ketamine cocktail solution (see below) using 0.4 mL/100 g body weight or Nembutal (0.1mL/100g).To prepare the ketamine cocktail solution:ketamine3.75 mlacepromazine0.30 mlrompun/xylazine1.90 mlsterile saline 23.0 ml Store in an airtight and light protected bottle for up to 3 months.2) Clear vasculature using Ringer's solution (37C):9.9 mL NaCl (79.8 g/L)1 mL KCl (37.5 g/L)1 mL Na2HPO4 (18 g/L)1 mL MgCl2 . 6 H20 (20.0g/L)2.5 mL NaHCO3 (50.0 g/L)Fill to 95.5 mL using ddH2O Warm to 37 C and bubble w/carbogen1 mL CaCl2 . 2 H2O (30.0 g/L)200 mg dextrose2.5 mL heparin1 mL xylocaine3) Perfuse with fixative for 6-15 minutes, depending on size of animal (6 minutes - mouse; 10 minutes - small rat; 15 minutes - large rat) using 4% paraformaldehyde in 0.1 M PBS, pH 7.4 (200 mL) at 37C:Heat 100 mL ddH2O to 60CAdd 8 g "Prill" paraformaldehydeAdd 4-6 drops 1 N NaOHFilter with #1 Whatman filterAdd 40 mL 5x PBSDilute to final volume of 200 mLAdd 0.1% glutaraldehyde if intending to photoconvert specimens. 4) Extract brain. If it is still soft, place in same fixative as above and allow to post-fix for 0.5-1 hour at 4C. Cut into 100-150m slices with Vibratome using a slow speed and the lowest frequency which will allow for proper cutting.5) Store slices in 0.1 M PBS in refrigerator. Slices should be used same day if planning to photoconvert. Given good initial fixation, slices will be usable for dye-filling for 2-3 days.6) Visualize tissue using IR-DIC Nomarski imaging. If necessary, counterstain tissue in 5 ?M acridine orange in 0.1 M PBS for 30 seconds. Microinject cells using 5% Lucifer Yellow-CH (lithium salt) in ddH2O. Use a positive retention current to prevent leakage, if necessary, and a negative pulsed current of 1-3 nA to inject cells. Allow cells to fill until all processes are equally fluorescent. 7) Place slices containing filled cells into 4% LY at 4C for at least 20-30 minutes. 8) Acquire light-level image of cell, using Mat-Tek dish to hold specimen. Mat-Tek dishes should be prepared ahead of time by treating with polyethyleneimine solution (0.1% aq.) for 30 sec., followed by brief rinse in ddH2O. Allow dish to dry and store in fridge until used. Can use PBS bubbled with Ar gas to try to reduce fading.9) Bubble DAB/potassium cyanide solution with O2: 2 mL DAB (10mg/mL)13 mg potassium cyanide2.66 mL 5x PBS 8.67 mL dd H2O9) Fix slice in 2% glutaraldehyde for 15-20 minutes:0.8 mL 25% glutaraldehyde2 mL 5x PBS7.2 mL dd H2O10) Wash 2-3 times in PBS.11) Place 100 mM glycine on slice for 1-2 minutes:38 mg glycine10 mL PBS12) Wash with PBS.13) Incubate slice in DAB/potassium cyanide solution for 8-10 min.14) Photo-oxidize slice with 75 W xenon lamp. Exchange DAB/potassium cyanide solution every couple of minutes. Allow reaction to proceed until all fluorescence is gone and brown reaction product is visible (about 10 minutes).15) Wash 3x 10 minutes in 0.1 M PBS at 4C.Conventional Embedding Procedure1) Post-fix in 0.5% OsO4 in 0.1 M PBS for 30 minutes at 4C.2) Rinse 3x 2 minutes in ddH2O at 4C.3) Dehydrate as follows: 70% EtOH, 10 minutes; 80% EtOH, 10 minutes; 90% EtOH, 10 minutes; 95% EtOH, 10 minutes; 100% EtOH, 2x 10 minutes; dry acetone, 2x 10 minutes (2nd time at room temp.)4) Infiltrate tissue in 50:50 acetone:DurcupanACM for 1 hour (or overnight).5) 100% resin for 2x 1 hour (2nd time fresh resin).6) Mount tissue on mould release slides and place in vacuum oven at 60C for 2 days.Microwave-Enhanced Embedding Procedure1) Osmium fix tissue in 600 L of 1% OsO4 for 2x 40 seconds. Solution should begin at < 10C and attain a final temp. of 30-35C. Microcentrifuge tube should not be placed in water bath during irradiation. 2) Rinse samples ddH20 for 2 minutes at room temp.3) Dehydration:50% EtOH2 X 40 sec.35C70% EtOH2 X 40 sec.35C90% EtOH2 X 40 sec.35C100% EtOH2 X 40 sec.35CDry Acetone2 X 40 sec.35CDehydration steps performed in water bath. Tubes should be filled with 600 L of solution for each step.4) Infiltration:1:1 Resin:acetone1 X 15 min.50C100% Resin3 X 10 min.50CFill bath with stock acetone and check level often to ensure that temp. probe is immersed in bath.5) Polymerization:Mount tissue on mould release slides and place in vacuum oven at 60C for 2 days.Obtain transmitted light z-series photo-oxidized, if desired, before sectioning.
Imaging product type
Type
Optical section
Cutting plane
transverse
Z resolution
0.5 um